Methotrexate (MTX) is a classical antifolate drug used for many years in the treatment of human cancers. This drug exerts its primary biological effect by inhibiting dihydrofolate reductase (DHFR), an enzyme which is responsible for the terminal step in converting the vitamin folic acid into a reduced form that actively participates in several crucial metabolic pathways within cells. The usefulness of MTX as a cancer chemotherapeutic, however, is often limited by intrinsic or acquired resistance of tumor cells to its effects, so that alternative antifolates effective against MTX-resistant tumors have been sought.
The potential therapeutic significance of folic acid analogues targeted against thymidylate synthase (TS) as opposed to DHFR was predicted more than 20 years ago by Borsa et al. (Cancer Res. 29:737, 1969). Shortly thereafter, the potent biological activity of 5,8-dideazafolic acid (1) was reported (Hutchison et al., Proc. Amer. Assoc. Cancer Res. 10:80, 1969; Bird et al., J. Mol. Pharmacol. 6:573, 1970). This led to an extensive program of synthesis of quinazoline analogues, and to the eventual selection of the N.sup.10 propargyl derivative 2 (CB3717, PDDF) as a suitable candidate for biochemical and clinical evaluation. While this compound has many desirable evaluation. While this compound has many desirable pharmacological characteristics, such as the ability to enter cells by a transport mechanism distinct from that of reduced folates and MTX, its clinical usefulness is hampered by hepatic and renal toxicities, which are due in part to low solubility at physiologic pH.
The toxicity problems encountered during clinical trials with CB3717 prompted a vigorous search for more soluble congeners, culminating in the discovery of a second-generation family of folate-based TS inhibitors, of which the prototypical example was 2-desamino-5,8-dideazafolic acid (3) and its N.sup.10 -methyl (4) and N.sup.10 -propargyl (5) analogues (Marsham et al., J. Med. Chem. 32:569, 1989; Jones et al., J. Med. Chem. 32:847, 1989). Also active were the corresponding 2-desamino-2-methyl analogues 6-8 and some of their congeners. ##STR2##
Biochemical studies with compounds 3-8 revealed that while replacement of the NH.sub.2 group at C.sup.2 by H or methyl generally results in weaker binding to purified TS in vitro, it also results in increased inhibitory potency against intact cells in culture (Jackman et al., Cancer Res. 50:5212, 1990; Jones et al.; Hughes et al., J. Med. Chem. observations are explained by the findings that (i) deletion of the 2-amino group does not diminish the analog's substrate activity for folylpoly-glutamate synthetase (FPGS), and (ii) polyglutamylation of the 2-desamino compounds increases binding to TS by as much as 100-fold (Moran et al., Mol. Pharmacol 36:7836, 1989; Jackman et al.). On this basis, a large number of congeners modified at C.sup.2, N.sup.10, and the aroyl moiety were synthesized to permit a detailed structure-activity analysis of 2-desamino-5,8-dideazafolates relative to the parent 2-amino compounds. The thiophene analogue 9 (ICI-D1694), with a methyl group at C.sup.2 and a methylated N.sup.10 emerged from these studies as the most promising desamino compounds for further evaluation (Jodrell et al., Proc. Amer. Assoc. Cancer Res. 31:341, 1990; Marsham et al., J. Med. Chem. 34:1594, 1991).